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*
Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, Oklahoma City, OK 73104;
Department of Microbiology and Immunology, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095; and
Joint Program in Neonatology, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A second aim of this study was to investigate the distribution of syndecan-4 in lymphohematopoietic tissues. Syndecans are highly regulated with respect to developmental expression and cell-type specificity as well as the extent and nature of glycosylation. An early finding was that syndecan-1 is present on pre-B cells, lost immediately before B cell maturation, and then reexpressed upon differentiation to the plasma cell stage (6). Northern blots of lymphoid tissues and transformed cell lines suggest that lymphocytes may express additional syndecans, including syndecan-4 (7). We have now explored this issue in greater detail with syndecan-4-specific mAbs and nontransformed cells. Our results revealed that syndecan-4 coincides with developmental progression of B lineage precursors. Ectopic expression and ligation of this glycoprotein caused dramatic morphological changes that may facilitate intercellular communication. HSPGs of this kind may contribute to formation as well as maintenance of the humoral immune system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and CB17 scid/scid mice were obtained from the Oklahoma Medical Research Foundation Laboratory Animal Resources Center. All experiments reported here were done with female mice at 4–8 wk of age. Wistar rats were purchased from Harlan (Indianapolis, IN).
Cell lines
B lineage cell lines (BCB10, BC7.12, 1A9, 70Z/3, WEHI231, CH12, BCL1, and SP2/0), T lineage cell lines (2B4 and BW5147), fibroblast cell lines (NIH 3T3 and L929), stromal cell lines (BMS2 and ST2), macrophge cell lines (J774A.1 and P388D1), a human renal carcinoma cell line transfected with large T Ag (293T), and the Jurkat human T lymphoma were maintained as previously described (5). The Ba/F3 and FDC-P1 immature hematopoietic cell lines were grown in medium conditioned by IL-3-transfected Chinese hamster ovary cells.
Transfection
Native murine syndecan-4 cDNA was inserted into the pEF-BOS vector (a kind gift from Dr. S. Nagata, Osaka Bioscience Institute, Osaka, Japan), and 2 µg of plasmid was then transfected into 293T cells using a calcium phosphate method. After 2 days of culture, 293T cells were detached with PBS containing 0.2 mM EDTA and stained with the KY series of mAbs. Truncated forms were also used that lacked the cytoplasmic domain of syndecan-4 and had the transmembrane domain from tissue factor with either the full extracellular or amino terminal portions of syndecan-4 as previously described (5). For stable transfection, the pRc/RSV vector containing the neomycin resistance gene was purchased from Invitrogen (Carlsbad, CA). Both plasmid DNAs were added to suspensions of either FDC-P1 or Ba/F3 cells in a cuvette (gene pulser cuvette; Bio-Rad Laboratories, Richmond, CA) and a 0.35 kV, 960 µF pulse was applied with a gene pulser (Bio-Rad). Transfected cells were cultured for 24 h and then selected in the presence of 1 mg/ml G418 (Sigma, St. Louis, MO). G418-resistant clones were selected for intensity of syndecan-4 expression by flow cytometry.
Expression of soluble recombinant Ig fusion proteins
A pEF-BOS-derived IgG1 expression plasmid was constructed
essentially as described (5). These constructs were introduced into
DH5
, expanded, and purified for transfection. Purified plasmids were
transfected into 293T cells with a calcium phosphate method. After 4
days of culture, supernatants were collected and used for
immunofluorescence staining or ELISA.
Preparation of mAbs
Wistar rats were immunized six times with BCB10, a pre-B cell
line found by RT-PCR to strongly express syndecan-4. Popliteal lymph
nodes were removed and fused with SP2/0 myeloma cells (American Type
Culture Collection, Manassas, VA). Over 2000 hybridoma supernatants
were screened by ELISA for reactivity to a syndecan-4 Ig fusion
protein. The resulting Abs (designated the KY series) were IgG2a,
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Abs were purified from the ascites fluid of CB17 scid/scid
mice that had been transplanted with these hybidomas.
Immunofluorescence staining
The fluorescent Abs used in these experiments were as follows.
The primary reagents FITC-conjugated anti-mouse CD3, CD19, CD24
(M1/69), BP-1, Thy1.2, Ly-6G (GR-1), PE-conjugated anti-mouse
TER119, CD5, CD43, and APC-conjugated CD45R (B220) were obtained from
PharMingen (San Diego, CA). PE-conjugated anti-mouse IgA was
obtained from Southern Biotechnology Associates (Birmingham, AL).
FITC-conjugated anti-Mac-1/CD11b was obtained from Boehringer
Mannheim Biochemicals (Indianapolis, IL). The new rat Ab against mouse
IL-7R (SB/199; IgG2b,) was established in our laboratory (Y.
Yamashita et al., manuscript in preparation). The secondary reagent
PE-streptavidin (Vector, Berlingame, CA) was used in dual staining
experiments, and RED613-streptavidin (Life Technologies, Gaithersburg,
MD) was added for three- or four-color staining. FITC-conjugated
anti-mouse IgM and FITC-avidin were obtained from Zymed (San
Francisco, CA)
To minimize nonspecific binding, cells were preincubated with the anti-Fc receptor mAb, 2.4G2 (American Type Culture Collection), and 10% normal rat serum for 20 min on ice and then washed. Cells were then incubated with the appropriate combinations of primary Abs in staining medium (PBS with 2% FCS, 0.1% NaN3) on ice for 20 min, washed twice with staining medium, then incubated an additional 20 min with PE-streptavidin (Vector) for dual staining and with RED613-streptavidin (Life Technologies) for three- or four-color staining experiments, and finally washed twice with staining medium. Flow cytometry analysis was conducted using a FACSCalibur (Becton Dickinson, San Jose, CA).
For detection of fusion protein binding, cells were stained with culture supernatants from 293T cells transfected with the F-syndecan-4-Ig/pEFBOS plasmid. FITC-conjugated goat anti-human IgG (Southern Biotechnology Associates) was used as a secondary Ab. Supernatants containing soluble CD44-Ig were used for negative controls. Staining efficiency was improved by inclusion of MnCl2 (5 mM) in the staining buffer (5) and by performing incubations at room temperature. Digestion with heparatinase (Seikagaku, Ijamsville, MD) was performed as described by Stanley et al. (8) except that the reactions were done in PBS containing 0.1% BSA.
ELISA
Capturing mAbs were coated onto 96-well microtiter plates, and fusion proteins were added. HRP-labeled goat anti-human IgG Ab (Southern Biotechnology Associates) was then used for detection of fusion proteins. Color was developed with a peroxidase substrate, and OD at 405 nm were measured on a microplate reader. The purified syndecan-1 ectodomain was prepared as described elsewhere (9). GST-fusion proteins of the syndecan-2–4 ectodomains were prepared by inserting the cDNA corresponding to the extracellular domain of each syndecan into the pGEX-2T expression vector (Pharmacia, Piscataway, NJ), in frame with the sequence encoding GST, expressing the vectors in Escherichia coli and purifying the fusion proteins by absorption and elution from glutathione Sepharose 4B (Pharmacia). The ectodomain and fusion proteins were coated onto 96-well microtiter plates, and rat mAbs were added. HRP-labeled goat anti-rat IgG (H+L) (Zymed) and peroxidase substrate (ABTS kit from Zymed) were then used for their detection. Results are presented as mean OD ± SD from triplicate wells.
Colony-forming cell assays
Murine bone marrow cell populations were suspended in 1 ml of assay medium as previously described (10). The semisolid agar cloning assay for B lymphocyte precursors was done with 2 ng recombinant mouse IL-7 (Upstate Biotechnology, Lake Placid, NY). The granulocyte-macrophage progenitor assay (CFU-c) was done with 25 µl of 10-fold concentrated L cell-conditioned medium as a source of CSF. All cloning assays were performed in 35-mm Petri dishes and were incubated at 37°C, 5% CO2. Colonies were scored on day 6. To separate syndecan-4 positive and negative populations from bone marrow, cells were stained with biotinylated KY/8.2 mAb followed by PE-streptavidin. After two washes, cells were sorted using the FACStarPlus cell sorter (Becton Dickinson).
Spreading assay and confocal microscopy
mAbs were bound to glass Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) by incubation at 4°C overnight at concentrations ranging from 10 µg/ml to 100 µg/ml. The wells were washed twice with HBSS (Life Technologies) and then blocked with 1% BSA or 10% FBS. Cells were plated on mAb-coated wells in culture medium and incubated for 4 h at 37°C. Photomicrographs were made after examination by phase contrast microscopy. Viable cells were stained with a monoclonal anti-syndecan-4 Ab different from that used for coating slides. The biotin-labeled primary reagent was revealed by staining with cychrome-labeled streptavidin, and the cells were washed and fixed with 3.7% formaldehyde plus 0.1% Triton X-100. Some of the fixed chamber slides were stained with PE-labeled phalloidin (Molecular Probes, Eugene, OR). Additional slides were stained with a purified polyclonal goat Ab to pp52, followed by FITC-labeled anti-goat IgG. After removal of chambers, slides were mounted with the Prolong anti-fade kit (Molecular Probes) and examined with a TCS-NT laser scanning confocal microscope (Leica, Heidelberg, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that an N-terminal fragment of syndecan-4, when expressed as a fusion protein with human IgG1, binds to a pre-B cell line (5). The full-length, native sequence of syndecan-4 has now been obtained and, with the exception of a 2-nt insertion in the noncoding region, was identical to one recently published (11). It has a high degree of homology with other members of the syndecan family and is very similar to the syndecan-4 characterized in other species (data not shown). Because the original fusion protein contained only the first 71 residues, we prepared a second fusion protein containing 122 aa to assess biological activity of the native syndecan-4 extracellular domain. We also conducted experiments to further define the nature of the syndecan-4 interaction with pre-B cells.
The full-length ectodomain recognized the BC7.12 pre-B cell line (Fig. 1
) in a manner indistinguishable from the
truncated version, demonstrating that the recognition portion of
syndecan-4 is located in the amino terminus and functional irrespective
of whether the membrane proximal domain is present. In addition to bone
marrow pre-B cells, syndecan-4-Ig bound to a small number of B cells in
spleen (not shown). Binding of the full-length domain was influenced by
divalent cations and dependent on HSPG modifications (Fig. 1). The
presence of 5 mM Mn2+ increased binding efficiency similar
to the 71-aa fragment (5). However, treatment with 5 mM EDTA, an amount
sufficient to totally block binding of the stromal interaction molecule
(SIM) fusion protein (5), only slightly interfered with syndecan-4-Ig.
Although the target of syndecan-4-Ig on pre-B cells is not known,
pretreatment of BC7.12 cells with heparatinase nearly abolished the
recognition reaction. Binding was also inhibited by the presence of
soluble heparin but not chondroitin sulfate A, pointing to the
involvement of HSPGs. Finally, the fusion protein was inactive when
prepared in 293T cells treated with sodium chlorate, an inhibitor of
sulfation (data not shown). These observations are consistent with the
heparan sulfate (HS) chains of syndecan-4 interacting with some HSPG on
pre-B cells.
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From a panel of mAbs generated against syndecan-4, two clones
(KY/8.2 and KY/103) that appeared to recognize distinct epitopes were
further characterized (Table I). The
KY/8.2 mAb had equal reactivity with both the truncated (71 aa) and
full-length (122 aa) ectodomain of syndecan-4 fusion proteins, whereas
KY/103 detected only the latter. These observations suggest that KY8.2
recognizes an epitope in the N-terminal portion while KY/103 targets
residues in the membrane proximal region. Both Abs specifically
recognized syndecan-4-GST fusion proteins prepared in bacteria and did
not bind to representatives of the other three members of the syndecan
family (Table II). These reagents also
stained syndecan-4-transfected 293T cells (data not shown). We conclude
that the Abs are specific to syndecan-4 and that posttranslational
modification of the protein core is not required for recognition.
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The new mAbs were used to investigate the distribution of
syndecan-4 on hematopoietic cells by multiparameter flow cytometry
(Fig. 2). Substantial numbers of
nucleated cells in the bone marrow were syndecan-4 positive.
Approximately 85% of the syndecan-4+ cells expressed the B
lymphocyte lineage marker CD19, although there was not complete
concordance between these two markers. Few, if any, syndecan-4-bearing
cells were myeloid (expressed either GR1/Ly-6G or Mac-1/CD11b), and
only a small fraction were in the erythroid lineage
(TER119+). Restriction of syndecan-4 expression to B cell
precursors suggested that the molecule might be acquired as a
consequence of lymphocyte maturation. Therefore, additional Abs were
used to resolve subsets of lymphocyte precursors (12).
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), but the marker was progressively
acquired by more mature cells. While only half of fraction C cells
(distinguished by BP-1 expression) were positive, this increased to
70% of pre-B cells (fraction D that lacks CD43; Fig. 4A) and nearly all
sIgM+ B cells (fractions E and F, distinguished by sIgM
densities) (Fig. 4, B and C).
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). This
might suggest a sequence through which IL-7-responsive lymphocyte
precursors acquire this glycoprotein. A subset of IL-7R+
cells is capable of responding to IL-7 in semisolid agar, and we used
cell sorting to assess their expression of syndecan-4 (Table III). Labeling of lymphocytes with the
syndecan-4-specific Abs did not influence their responsiveness to IL-7
(data not shown), and cells capable of IL-7-dependent clonal
proliferation were present in both syndecan-4+ and
syndecan-4- populations. In contrast, CSF-responsive
myeloid progenitors appeared to lack this marker and were enriched
among the syndecan-4- cells (Table III). Syndecan-4 was
not expressed on dendritic cells that were expanded by culture of bone
marrow cells for 1 wk with 20 ng/ml of GM-CSF or on myeloid cells grown
in long-term bone marrow cultures (data not shown).
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). Maturing lymphocyte precursors
expressed more syndecan-1 than syndecan-4, but the former is known to
be lost at the B cell stage and reacquired by Ab-secreting plasma cells
(6) (Table IV). While B lymphomas expressed syndecan-4, very little was
present on myeloma cells. As expected from our previous study and work
by others (3, 5), syndecan-4 was present on several stromal cell and
fibroblast lines. While the level of expression was always above
background and more remarkable with biotin-labeled Abs, it was less
than syndecan-1 on these cell types. Surface display of syndecans 1 and
4 was also demonstrated by immunofluorescent staining of viable primary
stromal cells in Whitlock-Witte cultures (not shown). We conclude that
syndecan-4 is predominantly associated with stromal cells and B
lymphocyte lineage precursors in bone marrow. Acquisition of the
syndecan-4 protein core on lymphocytes reflects their degree of
maturation, but does not precisely correspond to their ability to
respond to IL-7.
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In contrast to syndecan-1 (6), syndecan-4 is expressed by most
splenic B cells as well as an extremely small population of T cells
(Fig. 5). While over 90% of the
CD19+ or IgM+ B cells in spleen or lymph nodes
displayed syndecan-4, a distinct subset of B cells was always observed
to lack this protein. The small subset of splenic B cells that display
CD5 were not distinguished by absence of syndecan-4 (data not shown). A
more detailed analysis was made of CD5+ and
CD5- B cells in the peritoneal cavity (Fig. 6). While CD5+ B cells tended
to have a lower average density of syndecan-4, there was heterogeneity
among both subsets. Additional subsets of mature B cells were resolved
by four-color flow cytometry, and none of them correlated well with
syndecan-4- B cells (see Discussion).
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Syndecan-4 expression on B cells might be modulated as a consequence of, or in parallel with, Ag experience. Blast cells were generated in culture by stimulating resting splenic B cells with LPS, an Ab to CD40 or RP105. Each of these activated lymphocyte populations continued to display high densities of syndecan-4 (not shown). Furthermore, expression was more homogeneous than on corresponding control cells held in medium alone.
We then investigated syndecan-4 expression on B cells that have
undergone Ig isotype switching. Peyer’s patches are known to contain
substantial numbers of B cells that display IgA but have lost the
ability to synthesize IgM. These cells completely lacked syndecan-4
(Fig. 7A). In contrast,
IgM-bearing cells in this mucosal tissue displayed the same
heterogeneity in syndecan-4 density noted with splenic B cells (Fig. 7B). Similar to IgA+ cells, IgG+ B
cells in the spleen also lacked syndecan-4 (data not shown).
Collectively, these results indicate that B cells leave the bone marrow
bearing syndecan-4. Entry into a proliferative response increases
expression, but isotype-switched memory B cells may markedly
down-regulate syndecan-4.
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Syndecan-4 functions were tested by ectopic expression in two
early hemapoietic cell lines (FDCP-1 and BaF/3) demonstrated not to
bear this molecule (Table IV). Transfected cells expressed high levels
of syndecan-4 protein, with no apparent influence on growth rates, IL-3
dependence, or self-adhesiveness. The addition of soluble Abs to
syndecan-4 caused modest aggregation and did not abrogate their
requirement for growth factors. Thus, syndecan-4 made by these cells
did not mediate strong homophilic recognition.
A remarkable morphological change occurred when syndecan-4-transfected
cells contacted immobilized syndecan-4 Abs. In these experiments,
transfected cells were added to slides coated with anti-syndecan
mAbs as a mimic of potential syndecan-4 ligands. Within 4 h of
attachment, syndecan-4-transfected FDC-P1 cells developed numerous
long, thin filopodia (Fig. 8,
D and G) that frequently displayed antenary
branching. Nearly all of the transfected cells responded in this way,
but did not do so in synchronous fashion. No morphological changes were
observed when control rat IgG was used to coat slides (Fig. 8, E and H). The formation of dendritic processes
was more apparent in wells coated with KY/8.2 (N-terminal epitope) than
KY/103 (membrane proximal epitope), and in both cases was uniformly and
completely blocked by addition of soluble anti-syndecan mAbs or
soluble syndecan-4-Ig fusion proteins (data not shown). Neither the
transmembrane or cytoplasmic domains were required inasmuch as
constructs with the extracellular portion of syndecan-4 fused to the
transmembrane domain of tissue factor also mediated these dramatic
morphological changes (Fig. 8G).
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B). In contrast, syndecan-4
was more concentrated on the cell body (Fig. 9A). Therefore,
while syndecan-4 was not localized to the focal adhesions, ligation
through this molecule was associated with cytoskeletal reorganization
involving actin and pp52.
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Normal splenic B cells stimulated to form lymphoblast aggregates were subsequently placed on anti-syndecan-4-coated slides. Blasts induced by treatment with anti-CD40 developed dendritic processes similar to those described above for transfected cells except that the processes were less numerous and prominent. Approximately 70–80% of the blasts responded, suggesting that they were more heterogeneous than the syndecan-4-transfected cells described above. In contrast, cells stimulated with other activation signals including LPS or anti-RP105 did not undergo morphological changes even though they displayed high densities of syndecan-4 (data not shown). Indeed, RP105-activated B cells showed little tendency even to attach. These findings demonstrate that normal B cells can undergo dramatic shape changes on contact with ligands for syndecan-4, but the initial route of activation is important.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Syndecan-4 on stromal cells could mediate bidirectional communication
with B lymphocyte precursors. We demonstrate here that recognition of
pre-B cells occurs when the entire extracellular domain of syndecan-4
is expressed in a fusion protein and that this recognition is inhibited
by heparin, but not chondroitin sulfate A (Fig. 1). Heparitinase (HS
lyase I, specific for linkages in HS chains) treatment of pre-B cells
destroyed binding. Furthermore, the syndecan-4-Ig fusion protein lost
activity when prepared in sodium chlorate-treated cells, a technique
that reduces GAG sulfation (our unpublished observations). These
results would be compatible with the HS chains of syndecan-4
interacting with another HSPG on pre-B cells, and an obvious candidate
would be syndecan-4 itself.
A previous study demonstrated that the cellular environment can be critical for appropriate posttranslational modification and adhesive receptor properties of syndecans (16). The early progenitor (FDCP-1 and Ba/F3) and human T leukemia (Jurkat) or renal cell carcinoma (293T) cells used here may differ from normal B cell precursors with respect to protein glycanation. Indeed, the former two lines do not express HS on their surfaces (17). Therefore, it is uncertain whether syndecans, CD44, or other HSPGs on pre-B cells represent ligands for the syndecan-4 made by stromal cells. HSPGs are bound by a variety of lymphocyte-derived ligands. For example, CD45, a lymphocyte molecule not known to bear GAG chains, may recognize HSPGs on stromal cells (18). It is possible that CD45 binds to other HSPGs known to be made by stromal cells (3). The core protein of syndecan-4 has been shown to have a ligand on fibroblasts (19), so the range of possible syndecan-4-mediated interactions may be even greater than those revealed here.
Sanderson and colleagues found that syndecan-1 was present on pre-B cells, down-regulated at the mature B cell stage and reexpressed by plasma cells (6). Much broader expression of syndecan-4 was suggested by a more recent study, where Northern blotting revealed transcripts in a series of transformed cell lines (7). We used Abs specific for two syndecan-4 extracellular domains to document when this HSPG is acquired and lost by cells of the B lymphocyte lineage. Some fraction B cells (CD45R+ CD43+ CD24+ BP-1- cells) display syndecan-4, but the marker is not uniformly present on a slightly more mature category of bone marrow precursors (fraction C; CD45R+ CD43+ CD24+ BP-1+ cells). Many fraction B cells have undergone at least the first step (DH to JH) of Ig gene rearrangement and some precursors in this category proliferate in the presence of IL-7 (20, 21, 22, 23). While syndecan-4 initially appears during an early stage of B lymphocyte development, the onset of expression is not perfectly synchronized with other maturational changes. Expression of this protein may correspond to a previously unrecognized milestone in B lymphocyte lineage differentiation.
Heparitinase treatment of pre-B cells reduces their ability to bind and
respond to IL-7 (24). Therefore, syndecan-4, or another HSPG, acts as a
coreceptor for this cytokine, possibly in a manner similar to that
proposed for the basic fibroblast growth factor receptor (25). While
most syndecan-4+ pro-B cells express one component of IL-7R
(IL-7R), approximately half of the IL-7R+ cells lack
syndecan-4 (Fig. 3). This corresponds to the finding that nearly equal
numbers of IL-7-responding precursors were present in fractions sorted
on the basis of syndecan-4 expression (Table III). Similar results were
obtained by sorting on the basis of syndecan-1 expression (not shown).
It is possible that syndecan-negative precursors acquire this HSPG when
placed in culture. Thresholds of responsiveness of B lineage precursors
to IL-7 change with successful Ig gene rearrangement (49), potentially
a result of HSPG expression. In contrast to lymphocyte precursors,
myeloid progenitors in bone marrow did not express syndecan-4.
Syndecan-4 may be a unique marker for resolving mature B lymphocyte
subsets. Approximately 10% of B cells in spleen and lymph node lack
syndecan-4, and these cells do not closely correlate with previously
described populations. For example, the well studied "B1a"
lymphocytes are not discriminated from "conventional" B2 cells on
the basis of syndecan-4 expression (Fig. 6) and are less frequent in
spleen than syndecan-4-negative B cells (26). We found
syndecan-4-positive and syndecan-4-negative B cells among subsets that
were gated for CD21- CD23- (newly formed B
cells); CD21+ CD23- (marginal zone B cells);
or CD21+ CD23+ (follicular B cells) (27, 28, 29).
Syndecan-4- B cells had "mature" properties in that
they were IgMlow IgDhigh class
IIhigh and CD23+. They also included
CD24int as well as CD24high populations. Of
particular interest, the distinct subset of IgM-
IgA+ B cells in Peyer’s patches and the IgM-
IgG+ B cells in spleen uniformly lacked syndecan-4 (Fig. 7
and data not shown). Therefore, syndecan-4 might be down-regulated as B
cells emerge from immune responses as Ig isotype-switched memory cells.
Further study may reveal that many syndecan-4- B cells are
long lived and have other properties ascribed to memory cells.
Syndecan-4 is known to be actively shed from endothelial cells and this process is modulated by growth factors (30). It will be important to learn if the same is true for B lineage lymphocytes and stromal cells, because soluble syndecan-4 may condition the immediate microenvironment. As one example, syndecan-4 has recently been found to bind proteases and modify their interaction with anti-proteases (31). Interestingly, stromal cells and pre-B cells express several ectoproteases (32). Cell bound and/or soluble syndecans could modify their activity. Many other roles have been proposed for HSPGs, including their ability to serve as receptor components and docking sites for cytokines and chemokines (33). It was recently shown that chemokines are necessary for production of B lymphocytes as well as for organization of peripheral lymphoid tissues (34, 35). We have shown that syndecan-4 density changes with B lymphocyte lineage differentiation and is notably absent from isotype-switched B cells. It will be interesting to see if this correlates with changes in ability to capture and respond to a newly described, B cell-specific chemokine (36, 37).
There are now exceptions to the conventional view that B lymphocytes
have a strictly round morphology. Activated B cell blasts placed on
surfaces coated with Abs to several Ags generate extended filopodia
(38, 39, 40). In these circumstances, resting B cells were first exposed to
anti-Ig Ab in addition to IL-4. We found that Abs to syndecan-4
similarly trigger filopodia production by transfected FDCP-1 and Ba/F3
cells without cytokine stimulation (Fig. 8). Resting normal B cells had
to be previously activated for this response and the pathway of initial
B lymphocyte activation was important. B cell blasts prepared by
ligation of CD40 but not those prepared by stimulation via RP105 or LPS
made filopodia in response to subsequent contact with syndecan-4 Ab.
Memory B cells isolated from human tonsils have irregular outlines and
could represent in vivo homologues of the cells we generated (41). Such
extended dendritic processes could increase surface area and facilitate
physical contact between Ag-presenting B cells and other cells of the
immune system.
We found that pp52(LSP1) phosphoprotein was present throughout the filopodia of syndecan-4-ligated cells. Staining with phalloidin revealed colocalization of polymerized actin in these processes as expected because pp52 binds F-actin and is closely colocalized with F-actin in microfilament-rich cell surface projections on leukocytes (14, 15). Hairy cell leukemia provides an example where B lineage lymphocytes have an irregular outline (42), and levels of pp52(LSP1) have recently been found to correlate with the appearance of hairy cell projections.4 Furthermore, elevated pp52(LSP1) expression has been implicated in cytoskeleton-mediated morphological and functional defects in neutrophils (43).
Lebakken and Rapraeger observed morphological changes in lymphoblastoid cells that were transfected with syndecan-1 and exposed to surfaces coated with syndecan ligands or an Ab to syndecan-1 (44). Although spreading and lamellipodia formation were more prominent than in our experiments, they noted the appearance of filopodia with time in culture. While the cytoplasmic tail of syndecan-1 was not required, the authors concluded from cytochalasin D treatment that the response is dependent on transmission of signals via the cytoskeleton. Similarly, we found that morphological changes induced via syndecan-4 ligation were independent of the transmembrane and cytoplasmic domains.
Syndecan-4 is a conspicuous component of focal adhesion sites in
fibroblasts (45), and we found a very similar pattern of distribution
on hematopoiesis-supporting stromal cell lines (our unpublished
observations). However, the molecule was concentrated in the main cell
body of stimulated lymphocytes (Fig. 9). This would seem to be
inconsistent with the notion that syndecan-4-specific Abs exert
physical force on lymphocyte membranes, resulting in the dendritic-like
filopodia. Furthermore, an Ab to the amino-terminal portion of
syndecan-4 was more effective than one directed to a membrane-proximal
epitope, although the reagents stain syndecan-4 equally well. These
findings are consistent with syndecan-4 transmitting a signal for
morphological change. The cytoplasmic domain of syndecan-4 can
apparently associate with and activate protein kinase C (46), but the
transmembrane and cytoplasmic domains of syndecan-4 were not required
for the morphological response seen here. One possibility is that the
ectodomain of syndecan-4 can interact with other transmembrane-spanning
proteins to transmit signals. Of particular interest is the recent
finding that syntenin interacts with the cytoplasmic domains of
syndecans and overexpression of syntenin caused membrane extensions
(47). Cdc42 is a member of the Rho family of small GTP-binding proteins
(48). Over-expression of Cdc42 in macrophages results in filopodia
formation and a remarkably similar morphology to the activated B cells
described here (compare Fig. 4a of Ref. 48 to Fig. 6 in this
paper). It will be important to learn more about the biochemical basis
for filopodia formation in activated B lymphocytes.
The Abs we prepared recognize protein epitopes in two separate regions of the extracellular domain of syndecan-4. To date, we have found no biological activities influenced by these Abs other than the morphological change in B cells. For example, the Abs had no obvious effect on production of myeloid or lymphoid cells in long-term bone marrow cultures, the responsiveness of pre-B cells to IL-7, the activation of mature B cells by LPS, or by Abs directed to CD38, CD40, or RP105 Ags. Syndecan-4 functions might overlap with those of other HSPGs or our Abs might not be directed to critical portions of the syndecan-4 molecule. Regardless, this first study of syndecan-4 expression on normal, untransformed lymphocytes provides a basis for further investigation of HSPG-dependent biological processes in the immune system. Moreover, the observations suggest means through which syndecan-4 might facilitate intercellular communication between B lineage lymphocytes and surrounding cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, 825 N.E. 13th Street, Oklahoma City, OK 73104. E-mail address:
3 Abbreviations used in this paper: HSPG, heparan sulfate proteoglycans; HS, heparan sulfate; int, intermediate.
4 E. K. Miyoshi, P. L. Stewart, P. W. Kincade, M. B. Lee, R. Wall, and A. A. Thompson. Elevated pp52 (LSP1) expression and altered distribution correlates with the distinctive cell morphology of hairy cell leukemia. Submitted for publication.
Received for publication October 7, 1998. Accepted for publication February 19, 1999.
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increased the on rate of the interferon--interferon- receptor complex formation. J. Biol. Chem. 273:10919.This article has been cited by other articles:
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P. Vanlandschoot, F. Van Houtte, B. Serruys, and G. Leroux-Roels The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate J. Gen. Virol., January 1, 2005; 86(1): 75 - 84. [Abstract] [Full Text] [PDF]
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M. Zako, J. Dong, O. Goldberger, M. Bernfield, J. T. Gallagher, and J. A. Deakin Syndecan-1 and -4 Synthesized Simultaneously by Mouse Mammary Gland Epithelial Cells Bear Heparan Sulfate Chains That Are Apparently Structurally Indistinguishable J. Biol. Chem., April 4, 2003; 278(15): 13561 - 13569. [Abstract] [Full Text] [PDF]
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50% of B cells had acquired syndecan-4 by
fraction C (BP-1+). Note that virtually all
syndecan-4+ lymphocytes also express the IL-7R
